PROJECT SUMMARY After infection the demand for influenza (IAV) protein synthesis and processing, including formation of disulfide bonds in cysteines of IAV-proteins in the endoplasmic reticulum (ER) of lung epithelial cells induces ER stress. ER stress in turn induces an ER based unfolded protein response (UPR). Viruses activate and sustain UPR for their replication, however, chronic UPR results in pro-inflammatory response and induction of apoptosis. It is not clear whether specific UPR components are seized by IAV for propagation and they also contribute to massive increases in IAV-induced pro-inflammatory responses and injury to the lung. Our novel preliminary results suggest that IAV infection activates the latent transcription factor, ATF6?. ATF6? is known to bind to specific DNA elements of UPR related genes and upregulates their transcription. Computational analysis of IAV-induced cytokine/chemokine-promoters showed the presence of conserved ATF6? binding elements in the promoter regions of cytokines and chemokines. We next observed that deletion/inhibition of ATF6? decreases inflammation, cytokines/chemokines, IAV burden and airway hyperresponsiveness (AHR) in mice. Furthermore, we found that IAV infection increases specifically a PDI- PDIA3 in HBE cells and in mice infected with IAV. Lung epithelial specific ablation of PDIA3 significantly decreased disulfide bridges in IAV-proteins, cytokines/chemokines, IAV burden, and AHR responses in the lung. These results suggested a prominent role for ATF6? and PDIA3 during IAV infection and immunopathology. Our overarching hypothesis is that the IAV-induced activation of ATF6? and PDIA3 supports overt epithelial pro- inflammatory responses, disulfide bonds in IAV proteins and in pro-inflammatory cytokines to increase IAV propagation and lung immunopathology. We have designed two specific aims to test our hypothesis. In Specific Aim #1 we will determine the functional role of IAV-induced UPR-activated transcription factor ATF6? in regulating expression of cytokines/chemokines, subsequent induction of immunopathology and development of long lasting effects on lung health. The specific Aim #2 seeks to dissect the critical requirement of IAV-induced PDIA3 in disulfide mediated processing of IAV-proteins (HA & NA), pro-inflammatory cytokines/chemokines subsequent induction of immunopathology. In both aims we will use transgenic mouse models, cell culture and sensitive redox assays and biochemical assays. Most importantly we will assess the efficacy of specific inhibitors of ATF6? (Ceapin-A7) and PDIA3 (PACMA31) in easing IAV-induced UPR, decreasing subsequent pro- inflammatory responses, and ultimately resulting in resolution of IAV-induced immunopathology. These studies will shed light on the importance of the IAV-induced lung epithelial UPR in IAV propagation and overt pro- inflammatory responses and offers insight into new and highly needed treatment modalities for IAV infection and severe respiratory distress beyond supportive care.